In 1993 Victor Ambros and colleagues discovered the first small regulatory RNA, lin-4. Since that time and the subsequent discovery of RNA interference (RNAi) and small interfering RNAs (siRNAs) by Andy Fire, Craig Mello, and David Baulcombe, the small regulatory RNA field has expanded in directions no one could have predicted. We now know that most eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in a remarkably wide range of biological processes, including but not limited to: heterochromatin formation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. The mechanistic underpinnings of RNAi are broadly conserved across eukaryotes. dsRNAs are cleaved by Dicer-like enzymes into short 20-25 nucleotide RNAs 1. These small regulatory RNAs associate with a conserved family of proteins termed the Argonautes (Agos) 2. Together, Ago proteins, and their associated small RNAs, negatively regulate gene expression by recognizing and inhibiting complementary nucleic acids. Our long-term goal is to understand the how and why of small RNAs and RNAi. Towards this goal we have conducted the first forward genetic screen in metazoans seeking to identify factors required for small RNA- mediated silencing in the nucleus (nuclear RNAi). To date our screen has identified two evolutionarily conserved factors, nuclear RNAi defective-2 (nrde-2) and nrde-3, which are required for nuclear RNAi. nrde-3 encodes an Ago protein that transports siRNAs from the cytoplasm to the nucleus 3. nrde-2 encodes an evolutionarily conserved protein that associates with NRDE-3 in the nucleus and is directed by NRDE-3/siRNA ribonucleoprotein complexes to nascent transcripts that have been targeted by RNAi. Our data indicate that small RNAs, acting in conjunction with NRDE-2 and NRDE-3, direct a novel mode of gene regulation: termination of RNA Polymerase II transcription. These data suggest that our genetic screen is targeting a dedicated nuclear silencing pathway and hint that the nuclear silencing pathway we are defining represents a novel and conserved mode of gene regulation. The experiments we propose in this grant are designed to identify and characterize additional components of the nuclear RNAi pathway, unravel the mechanism of nuclear RNAi, and begin to elucidate the raison d'jtre of small RNA-mediated gene silencing in metazoan nuclei. These experiments will provide a framework for us, and others, to ask if these processes are mechanistically conserved in mammals. Initial successes utilizing siRNAs to target oncogenic and viral mRNAs have generated excitement that siRNAs may be utilized eventually to treat human disease 4. siRNA treatment could theoretically be used to down- regulate any RNA molecule, either foreign or endogenous, present within the human body. Prior to the rational use of siRNAs in the treatment of human disease, however, it is essential that we understand: how small RNAs are generated, how and where small RNAs function, the specificity of small RNA-driven silencing, and the role of small RNAs in endogenous biological processes. Our research is addressing these questions. PUBLIC HEALTH RELEVANCE: siRNA therapeutics may eventually be used to regulate gene expression in humans. Prior to the rational use of siRNAs in the treatment of human disease, however, it is essential that we understand: how small RNAs are generated, how and where small RNAs function, the specificity of small RNA-driven silencing, and the role of small RNAs in endogenous biological processes. Our research is addressing these questions.